The present invention relates to methods of using low-strength electric fields to treat tumors. More particularly, the present invention relates to methods and an apparatus which utilize low strength pulsed electric fields on the order of 20-70 V/cm, with or without adjunct chemotherapy to treat or cure various tumor and cancerous tissue.
Cancer is second only to heart disease as a cause of death, accounting for 22% of all deaths (Fraumeni J F, Devesa S S, Hoover R N, Kinlien L J. Epidemiology of cancer. In: Cancer—principles and practice of oncology, DeVita V T, Hellman S, Rozenberg S A, (eds.) pp. 150, Lippincott J. R. Co., Philadelphia, 1993. Colon cancer, melanoma and breast cancer are three particularly problematic types of cancer.
Melanomas are aggressive, frequently metastatic tumors derived from either melanocytes or melanocyte related nevus cells (“Cellular and Molecular Immunology” (1991) (eds) Abbas A. K., Lechtman, A. H., Pober, J. S.; W.B. Saunders Company, Philadelphia: pages 340-341) which make up approximately 3% of all skin cancers. Of particular concern is the current worldwide increase in melanoma which is unsurpassed by any other neoplasm with the exception of lung cancer in women (“Cellular and Molecular Immunology” (1991) (eds) Abbas, A. K., Lechtiman, A. H., Pober, J. S.; W.B. Saunders Company Philadelphia pages: 340-342; Kirkwood and Agarwala (1993) Principles and Practice of Oncology 7:1-16). The aggressiveness of melanoma is such that even when melanoma is apparently localized to the skin, up to 30% of the patients will develop systemic metastasis and the majority will die.
Breast cancer is a significant health problem for women in the United States and throughout the world. Although advances have been made in detection and treatment of the disease breast cancer remains the second leading cause of cancer-related deaths in women, affecting more than 180,000 women in the United States each year. For women in North America, the life-time odds of getting breast cancer are now one in eight.
Colon cancer is the second most frequently diagnosed malignancy in the United States as well as the second most common cause of cancer death. About 100,000 new cases of colon cancer are diagnosed yearly, with about 50,000 deaths. The five-year survival rate for patients with colorectal cancer detected in an early localized stage is 92%; unfortunately, only 37% of colorectal cancer is diagnosed at this stage. The survival rate drops to 64% if the cancer is allowed to spread to adjacent organs or lymph nodes, and to 7% in patients with distant metastases. Recurrence following surgery (the most common form of therapy) is a major problem and is often the ultimate cause of death. In spite of considerable research into therapies for the disease, colon cancer remains difficult to diagnose and treat.
The leading cause of cancer death in general is due to growth of metastases since, in the majority of cases, by the time a malignancy has been diagnosed, metastases have already spread to other sites (for review see Fidler and Balch, 1987. Curr. Prob. Surg. 24:137). Whereas metastatic primary tumors can in many cases be surgically removed, greatly contributing to satisfactory therapeutic outcomes, metastases, such as disseminated micrometastases, can be difficult or impossible to locate and/or reach and thus surgical removal such metastases is usually not an option. Thus, metastases pose the most serious challenge to cancer therapy and are the main cause of failure of treatment of this disease. Therefore, prevention of metastasis is necessary to improve the prognosis of cancer patients.
In order to treat cancer effectively efficient removal of the primary tumor mass and prevention of secondary tumor growth, and eradication of metastatic cells must be achieved. Furthermore, significant prevention of recurrence of cancer growth can be achieved by generation of anti-cancer immune responses.
Surgical excision of tumors is the most widely employed therapeutic modality for the treatment of cancer, in which the primary goal is the complete eradication of local and regional tumor. This involves removal of adequate margins of normal tissue surrounding the tumor, and radical wide excision in order to prevent local recurrence. However, despite major advances in the surgical pre- and postoperative care of patients, surgical treatment of malignant neoplasms remains highly limited (Eilber F R. Principles of cancer surgery. In: Cancer Treatment, Haskell C M, (ed.) 5th ed., pp. 47, W.B. Saunders Co. Philadelphia, 2001). Surgical techniques are effective only in the area of the primary tumor or regional lymphatics and do not affect neoplasms located outside the operative field. Furthermore, due to anatomic location, many tumors cannot be treated by surgical resection because removal of an adequate margin of normal tissue cannot be achieved. Also, surgical treatment is often not an option for tumors intimately involving major blood vessels or essential organs. As well, many patient present problematic medical histories, such as cerebrovascular or cardiovascular accidents, or uncontrolled diabetes, rendering them poor surgical candidates because of their high postoperative mortality rate. Also, in many cases, tumor excision can not performed without causing unacceptable levels of impairment of physiologic functions or cosmetic damage.
Chemotherapy alone or in combination with surgery is commonly the most efficient anti-cancer remedy (Haskell C M. Principles of cancer chemotherapy. In: Cancer Treatment, Haskell C M, (ed.) 5th ed. pp. 62-86, W.B. Saunders Co. Philadelphia, 2001). However, chemotherapeutic agents often cause severe and unacceptable side-effects, such as bone marrow and lymphoid organ damage resulting in immunosuppression, thereby rendering subjects highly vulnerable to lethal opportunistic infections, as well as various other types of organ toxicities. Thus, the use of cytotoxic drugs is limited only to tolerated doses. One way to reduce minimal therapeutic doses of chemotherapeutic agents would be to enhance the efficiency of uptake of chemotherapeutic drugs into cancer cells.
During the last two decades, various techniques based on biological, chemical and physical processes have been developed for facilitating incorporation of macromolecules into cells. Methods for intracellular delivery of exogenous substances based on biological phenomena have employed molecules controlling the activity of specific membrane channels in various cell types (Heppel and Weisman, 1985. J Membr Biol. 86:189), pore-forming toxins (Ahnert-Higler et al., 1989. Methods Cell Biology 31:63) and liposome-endocytosis mediated delivery of compounds (Friend et al., 1996. Biophys Acta 1278:41). Some permeabilization methods are based on chemical modification of cell membranes by various substances, most commonly via the use of detergents as permeabilizing agents. Other methods based on chemically induced permeabilization include protease digestion or stimulation of DNA binding to the cell surface by formation of neutral complexes of DNA with various molecules. Physical methods of introducing molecules into cells include application of hypotonic stress (Poulin et al., 1993. J Biol Chem. 268:4690), cell bombardment by coated molecules (Salford et al., 1993a), microinjection (Soreg and Seidman, 1992. Methods Enzymol. 207:225), electroporation (Potter, 1993. Methods Enzymol. 217:461), and exposure to pulsed low electric fields (LEFs) (Rosenberg and Korenstein, 1997. Bioelectrochemistry and Bioenergetics 42:275).
Electroporation involves formation of a reversible, high permeability plasma membrane state in cells or bacteria exposed to 50-200 μs pulses of high-strength electric fields in the range of 500-5000 V/cm. At a threshold value of about 1 V across the cell membrane, a sudden increase in membrane permeability is observed which is thought to be mediated by stabilization of transient membrane defects and to their expansion to large metastable hydrophilic pores. Both transient and stable pores can be the sites of extrinsic material entry into the cell (Hapala, 1997. Crit Rev Biotechnol. 17:105; Rols and Teissie, 1990. Biophys J. 58:1089; Rols and Teissie, 1998. Biophys J. 75:1415). Electroporation also appears to involve stimulation of biological endocytosis in areas of destabilized membrane structure (Rols et al., 1995. Biochem Biophys Acta 1111:45). While electroporation, also termed electropermeabilization or electroinjection, has been generally used as a method of transfecting cells with nucleic acids, this method has also been used to load cells with a variety of other molecules, including proteins (Lambert et al., 1990. Biochem Cell Biol. 68:729), such as phalloidin (Hashimoto et al., 1989. J Biochem Biophys Methods 19:143) or antibodies (Chakrabarti et al., 1989. J Biol Chem. 264:15494).
In contrast to electroporation, incorporation of macromolecules into cells via exposure to LEFs, a methodology developed by the present inventors, utilizes low voltage electric fields. Exposure of cells and vesicles to LEFs leads to efficient intracellular incorporation of various molecules, including carbohydrates, such as polysaccharides, and proteins, such as β-galactosidase (Rosenberg and Korenstein, 1997. Bioelectrochemistry and Bioenergetics 42:275) via an underlying mechanism involving endocytosis-like processes. Exposure of membrane vesicles and cells to such LEFs leads to electrophoretic lateral mobility of charged proteins and lipids in the plane of the cell membrane (Poo, 1981. Bioeng. 10:245; Brumfield et al., 1989 Biophys J. 56:607), and generation of transmembrane potential differences (Farkas et al., 1984. Biophys J. 45:363). It has been shown by the present inventors that exposure of cells in suspension or monolayer to trains of pulsed unipolar electric fields in the range of about 1-100 V/cm (Rosenberg and Korenstein, 1997. Bioelectrochemistry and Bioenergetics 42:275), or to AC fields with peak-to-peak amplitudes of about 1-60 V/cm leads to efficient uptake of macromolecules having molecular weights ranging from about 1 to 2000 kDa, an exceptionally broad range. Unlike following electroporation, cells exposed to LEFs in vitro maintain high viability due to the magnitudes of the applied electric fields being too low to induce changes in membrane permeability via physical disruption of its integrity (Rosenberg and Korenstein, 1990. Biophys J. 58:823). LEFs have been shown to induce endocytosis, a process which includes a complex sequence of membrane-linked processes resulting in uptake of extrinsic substances involving binding of such substances to the cell surface, formation of endocytotic vesicles and maturation of endocytotic vesicles to lysosomes (Mellman, 1996. Ann Rev Cell Dev Biol. 12:575).
Several prior art approaches employing electric fields have been employed to treat tumors.
One approach has employed electroporation in conjunction with netropsin, bleomycin, or melphalan to attempt to increase of the cytotoxicity of these drugs against cultured DC-3F cells (Orlowski S. et al., 1988. Biochem Pharmacol. 37:4727).
Yet an additional approach has utilized electroporation in conjunction with daunorubicin, doxorubicin, etoposide, paclitaxel, carboplatin or cisplatin in order to attempt to potentiate their cytotoxic effect against cultured cells (Gehl J. et al., 1998. Anticancer Drugs 9:319).
Still another approach has employed very high strength electric field electroporation (1,300 V/cm) in conjunction with administration of cis-diamminedichloroplatinum (II) in order to attempt to treat SA-1, EAT, or B16 melanoma tumors in mice (Sersa G. et al., 1995. Cancer Res. 55:3450)
An additional approach has used electroporation in conjunction with administration of bleomycin in order to attempt to treat tumors of the female genital squamous cell carcinoma cell line CaSki in nude mice (Yabushita H. et al., 1997. Gynecol Oncol. 65:297).
Another approach has used very high strength electric field electroporation (1,300 V/cm) in conjunction with administration of bleomycin to attempt to treat head and neck squamous cell carcinoma (Belehradek J J. et al., 1993. Cancer 72:3694).
Yet another approach has utilized very high strength electric field electroporation (1,000-1,300 V/cm) in conjunction with administration of bleomycin to attempt to treat head and neck squamous cell carcinoma, and salivary and breast adenocarcinomas (Domenge C. et al., 1996. Cancer 77:956) However, all of the aforementioned prior art approaches suffer from significant disadvantages.
For example, all of these prior art approaches have employed electroporation, and hence high-strength electrical fields which, as described hereinabove, are significantly cytotoxic and which, by their extreme nature, are inherently hazardous. Electroporation suffers from the drawbacks of being inefficient in its potentiation of drug uptake, and in being restricted with respect to the range of molecular weights of the molecules whose uptake it is capable of potentiating. Prior art approaches have demonstrated potentiation of the in vivo anti-tumor effect of a restricted number of chemotherapeutic drugs (bleomycin, cisplatin, adriamycin and 5-fluorouracil). Moreover, none of these prior art methods has been shown to be effective in curing cancer at a metastatic stage. Critically, none of these prior art approaches has been shown to be effective against tumor cells in the absence of chemotherapeutic agents. Also none of these prior art approaches has demonstrated the capacity to upregulate anti-tumor immune responses as demonstrated by resistance to challenge.
Thus, all prior art approaches have failed to provide an adequate solution for treating tumors using electrical fields.
There is thus a widely recognized need for, and it would be highly advantageous to have, a method and apparatus devoid of the above limitation.